Immuno-specificity Of Multi-determinat Artificial Antigen And Gold Immuno-chromatography Assay For Multi-residue Of Pesticide

Haptens of chlorpyrifos(CHBu),Triazophos(THHe),carbofrua(BFNB)and parathion-methyl(MP5)were synthesized in this study.Four kinds of multi-determinant artificial antigen was prepared by those haptens conjugating to the carrier protein(BSA,HSA,KLH)in turn.Male New Zealand white rabbits were immunized with those multi-determinant immunogen separately or mixed to produce the polyclonal antibodies(PAbs),which can recognize the four pesticides.Four kinds of broad specificity PAbs which we obtained displayed high level for each relative hapten-OVA conjugate.Characterization studies of those PAbs showed that they have acceptable affinity and specificity to the four relative pesticides.An indirect competitive ELISA was developed for multi-residue determination.The I₅₀ range of four PAbs for the four pesticides was 0.0617-1.2208μg/mL,0.0242-0.1388μg/mL,0.4567-30.6194μg/mL and 0.1156-7.5054μg/mL,with the detection limit(I₁₀) range of 0.0024-0.0451μg/mL,0.0001-0.0013μg/mL,0.0062-0.0516μg/mL and 0.0019-0.0211μg/mL for carbofuran,triazophos,chlorpyrifos and parathion methyl, respectively.Results indicated that,this study provided a new strategy to develop immunoassays through artificial antigen design for pesticides multi-residue determination.The mixture immunogen(CHBu-BSA,THHe-BSA,BFNB-BSA and MP5-BSA, 1:1:1:1:1,m/m)were also immunized to rabbits as controls.Relevant broad specificity PAbs with high titer was obtained.Direct and indirect competitive ELISA method was used to study the characterization of this PAbs,obviously inhibitions of four pesticides was not found,and the ELISA curves therefore could not established either.Four kinds of monoclonal antibodies(McAbs)derived from BFNB-BSA,THHeBSA, CHBu-BSA and MP5-BSA respectively,were obtained.The I₅₀value for the four pesticides was 0.1356,3.4086,8.3890 and 3.2045 ng/mL,with the detection limit (I₁₀)of 0.0207,0.2325,0.7556 and 0.4004ng/mL for triazophos,carbofuran, chlorpyrifos and parathion methyl,respectively. Gold immuno-chromatography assay(GICA)based on nanocolloidal gold-labeled antibody or antigen for detection of multi-residue of pesticides was developed.The nanocolloidal gold,with an average particle diameter of 25 nm(G25)was labeled to broad specificity PAbs,McAbs,bispecific-McAb(bis- McAb)and OVA-hapten conjugate respectively.After comditions optimization,two kinds of strips were finally developed for the residue determination of triazophos and carbofuran.The limit of detection(LOD)for triazophos and carbofuran of the strip were 0.004μg/mL and 0.031μg/mL which based on gold-labeled McAbs,and were 0.008μg/mL and 0.063μg/mL which based on gold-labeled bis- McAb,respectively.